Abstract: Background and Objective: Infections due to Salmonella serovars represent a significant public health risk and are economically important for the poultry industry. Genes involved in pathogenesis of Salmonella serovar are clustered within Salmonella pathogenicity islands. Meanwhile, over use of antibiotics in poultry farms has led to an increase in antibiotic resistant Salmonella strains, which can be challenging to control. The present study was conducted to determine antibiotic resistance profiles and to detect the presence of five major pathogenicity islands among Salmonella serovars isolated from chickens in Egypt. Materials and Methods: Samples (n = 930) taken from chicken hearts, livers, caeca, yolk sacs, ovaries and cloacal swabs were collected and used for isolation and serotyping of Salmonella species. Antibiotic resistance was determined using the antibiogram method. The PCR was used for the molecular detection of Salmonella species using primers targeting invA and the pathogenicity islands genes invaE/A, ssaQ, mgtC, spidR and sopB, which are found in the most prevalent Salmonella serovars that present public health concerns. Results: The detection of 30 Salmonella isolates was confirmed by conventional and PCR methods and additional 5 Salmonella isolates were detected only by PCR. Among the isolates, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Muenster, Salmonella Anatum and Salmonella Virchow were the most prevalent serotypes at 36.7, 26.7, 20, 10 and 6.6%, respectively. The Salmonella serovars showed three antibiotic susceptibility patterns and all prevalent serovars carried the five virulence genes. Conclusion: Salmonella serovars that are pathogenic in chickens and that have public health relevance, including Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Muenster, are circulating in chicken farms in Egypt.
INTRODUCTION
Salmonella infection can cause severe economic losses for the poultry industry1. Chickens can be infected with many different serovars of paratyphoid Salmonella, including Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Heidelberg, which are avian pathogens that exist worldwide2.
Salmonella is the most common etiological agent of foodborne diarrheal illness3-5. Thus, detection of Salmonella in primary poultry production is an issue of interest since control of this zoonotic disease is mainly based on restricting pathogen distribution on chicken farms6. Furthermore, there is increasing concern about Salmonella pathogens due to increasing spread of antibiotic resistance and evolution of more pathogenic strains7,8. The inappropriate use of antibiotics on chicken farms in developing countries, including Egypt, is thought to be a main reason for the increasing the frequency of multidrug resistant Salmonella9. Multidrug resistant Salmonella serovars include Salmonella Typhimurium and Salmonella Enteritidis, which have been able to infect humans and cause systemic infection and death due to treatment failure10.
The outer proteins of Salmonella spp. (SOPs) contribute to invasion by these bacteria through the compromise of membrane integrity11 and cytoskeletal alterations in host cells12. Meanwhile, Salmonella spp. pathogenicity islands (SPIs) are of a critical importance for Salmonella virulence, as they encode a molecular apparatus called the type III secretion system (TTSS) that injects bacterial effector proteins through bacterial and host membranes to interact with host cells13. The ability of Salmonella to efficiently colonize host cells is attributed to gene clusters, including SPIs, which encode virulence factors that are distributed in the Salmonella genome2. Several major Salmonella pathogenicity islands have been reported for different serovars, SPI-1-5 is the predominant type in most serovars whereas, others are less widely distributed2,13,14. In general, SPI-1 is responsible for the invasion of host cells and induction of macrophage apoptosis, SPI-2 contributes to systemic infection and replication within macrophages, SPI-3 is required for bacterial survival in macrophages and for Salmonella growth in low magnesium environments, SPI-4 is essential for intra-macrophage survival and harbors genes that are important for toxin secretion and apoptosis, whereas, SP-5 encodes genes for multiple Type III secretion system effector proteins2,13-19.
In this study, it is reported that the isolation, identification, serotyping and antibiotic resistance patterns of Salmonella spp. are isolated from chickens in Upper Egypt. In addition, the frequency of virulence-associated genes in isolates was assessed, particularly for those genes that have zoonotic importance.
MATERIALS AND METHODS
Sample collection: A total of 240 diseased and freshly slaughtered chickens (155 baby chicks, 70 broilers and 15 layers) were subjected to post-mortem examination were used in this trial. A total of 930 samples from livers, hearts, caeca and yolk sacs (baby chicks only) as well as ovaries and cloacal swabs (layer flocks only) were collected from the birds under completely aseptic conditions.
Isolation and identification of Salmonella isolates: The Salmonella isolation and identification processes were carried out according to ISO 657920. In brief, the samples were directly inoculated in buffer peptone water (Oxide) and incubated at 37°C for 18 h as a pre-enrichment step. Then, 100 μL of culture was transferred into a tube containing selenite cysteine broth (Oxide) and incubated at 37°C for 24 h before a loopful of bacterial from selective enriched media was streaked onto MacConkey’s agar (Oxide), Salmonella Shigella agar (Oxide), Bismuth sulfate agar (Oxide) and Xylose-lysine deoxycholate (XLD) agar (Oxide) and incubated at 37°C for 24 h. The suspected Salmonella colonies were identified by Gram staining and motility tests. Biochemical assays included triple sugar iron (TSI) agar (Oxide), urease agar (Oxide) and citrate agar (Oxide) according to ISO 657920.
Serotyping of Salmonella isolates: Isolates that were biochemically identified as Salmonella spp. were serotyped using O and H antisera (Difco, Detroit, USA) by a slide agglutination test, according to the Kauffmann-White classification scheme21.
Determination of the antibiotic susceptibility profile of Salmonella serovars: Strains were tested for antibiotic resistance by the plate disc diffusion method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines22. The following discs were included in the test: Ciprofloxacin (CIP), 5 mg (Bioanalyses), levofloxacin (LEV), 5 mg (Bioanalyses), norfloxacin (NOR), 10 mg (Oxide), chloramphenicol (C), 30 mg (Bioanalyses), amoxicillin/clavulanic acid (AMC), 30 mg (Bioanalyses), streptomycin (S), 10 mg (Bioanalyses), trimethoprim (TMP), 5 mg (Bioanalyses), doxycycline (DO), 30 mg (Oxide), cephradine (CE), 30 mg (Oxide), rifampicin (RD), 5 mg (Oxide) and lincomycin (L), 2 mg (Oxide).
| Table 1: | Oligonucleotide sequences of primers used to detect Salmonella species and virulence associated genes in Salmonella isolates |
The results were interpreted as recommended by the CLSI22 to determine if the strain was resistant, intermediate or susceptible to the tested antibiotics.
Molecular detection of Salmonella species and five major virulence associated genes by PCR: The virulence genes invaE/A, ssaQ, mgtC, spidR and sopB were detected as described by Soto et al.23 and Sanchez-Jimenez et al.24. The DNA was extracted from the cultures using a QIAamp DNA mini Kit (Qiagen, USA) according to the manufacturer’s instructions. The PCR was performed in volume of 25 μL containing: 12.5 μL GoTaq® Hot Start Green Master mix (Promega, USA), 1 μL 20 μM each forward and reverse primer (Table 1), 3 μL DNA template and 7.5 μL nuclease-free H2O. The PCR was carried out using a gradient thermal cycler (A200, gradient thermal cycler, Japan) under the following conditions: Initial denaturation at 95°C/2 min followed by 30 cycles of denaturation at 95°C/1 min, annealing at 51°C/1 min (invaE/A and spidR), 53°C/1 min (sopB), 54°C/1 min (mgtC) or 58°C/1 min (ssaQ) and extension at 72°C/1 min before a final extension at 72°C/5 min. A total of 15 μL of PCR product in 3 μL loading buffer was loaded on a 1.5% agarose gel and electrophoresed at 100 volts/35 min before staining with 0.5 μg ethidium bromide/1 mL of TAE running buffer for 30 min and visualization under a UV illuminator.
RESULTS
Incidence of Salmonella serovars isolated from chicken flocks: Out of the 240 chickens in the sample, 30 were positive for Salmonella (incidence of 12.5%) as indicated by conventional bacteriological methods using MacConkey’s agar, Bismuth sulfite agar, Salmonella Shigella agar and XLD media (Table 2). All suspected Salmonella isolates were Gram negative, straight, non-spore forming rods by Gram staining of colonies. Furthermore, Gram staining showed Gram negative bacilli and a motility test indicated that the suspected isolates were highly motile. Biochemically, all suspected isolates produced alkaline (red) slant and acid (yellow) butt with or without H2S production on TSI and were urease negative and citrate positive (Table 2).
Serotyping of Salmonella isolates: The 30 Salmonella isolates were serotyped using O and H antisera (Table 2). The recovered serotypes were 11 (36.7%) Salmonella Enteritidis, 8 (26.7%) Salmonella Typhimurium, 6 (20%) Salmonella Muenster, 3 (10%) Salmonella Anatum and 2 (6.6%) Salmonella Virchow.
Antibiotic susceptibility profile of isolated Salmonella serovars: An antibiogram of the isolated Salmonella serovars was compiled using 12 antibiotic discs to determine the most suitable antibiotic to control Salmonella infections (Table 3). The antibiogram showed three antibiotic susceptibility patterns with multidrug resistance: (i) Type I multidrug resistant to cephradine, rifampicin and lincomycin, including Salmonella Enteritidis and Salmonella Virchow isolates, (ii) Type II multidrug resistant to doxycycline, cephradine, rifampicin and lincomycin, including Salmonella Typhimurium and Salmonella Muenster isolates and (iii) Type III multidrug resistant to streptomycin, trimethoprim, doxycycline, cephradine, rifampicin and lincomycin, seen for Salmonella Anatum (Table 3). All identified Salmonella serotypes were 100% sensitive to ciprofloxacin, levofloxacin, norfloxacin, amoxicillin/clavulanic acid and chloramphenicol, 80% sensitive to streptomycin and trimethoprim, 100% resistant to cephradine, lincomycin and rifampicin and 60% to doxycycline.
Molecular identification and characterization of pathogenicity islands in Salmonella serovars: Among the 240 chickens tested with PCR and primers targeting a 284 bp region of invA as a Salmonella specific gene, the Salmonella prevalence was 14.6% (35 out of 240).
| Fig. 1: | PCR products of five pathogenicity island genes (SP-1 (450 bp), SP-2 (677 bp), SP-3 (655 bp), SP-4 (1269 bp) and SP-5 (1170 bp)) from the most prevalent Salmonella spp. that are a public health concern. The PCR products were resolved on a 1.5% agarose gel stained with ethidium bromide (0.5 μg mL–1) after electrophoresis and photographed using a PhotoDoc-It-Imaging gel documentation system (Ultra-Violet Products Ltd., UK) equipped with a Canon digital camera |
Lane M: 100 bp DNA ladder (Solis BioDyne, Estonia), Lanes (1-5): Pathogenicity islands of Salmonella Enteritidis, Lanes (6-10): Pathogenicity islands of Salmonella Typhimurium, Lanes (11-15): Pathogenicity islands of Salmonella Muenster, NC: Negative control (PCR master mix without template DNA) |
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| Table 2: | Serotyping of Salmonella spp. isolated from chickens |
| Table 3: | Antibiotic susceptibility of Salmonella serovars isolated from chickens |
AMC: Amoxicillin/clavulanic acid, C: Chloramphenicol, CE: Cephradine, CIP: Ciprofloxacin (CIP), DO: Doxycycline, L: Lincomycin, LEV: Levofloxacin, NOR: Norfloxacin, RD: Rifampicin, S: Streptomycin, TMP: Trimethoprim |
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All 30 positive isolates were identified biochemically and serologically as Salmonella spp., in addition to the 5 suspected samples that were atypical Salmonellae according to biochemical and serological methods.
The three highly prevalent Salmonella serotypes (Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Muenster) were investigated for the presence of the five major SPIs, SPI-1, SPI-2, SPI-3, SPI-4 and SPI-5 by conventional PCR using gene sequence-specific primers. The results indicated that the five pathogenicity islands encoding invE/A, ssaQ, mgtC, spidR and sopB were found in the three predominant Salmonella serovars (Fig. 1).
DISCUSSION
Salmonella infections not only adversely affect public health and the poultry industry but surveillance, treatment and prevention of infections by these bacteria can be costly and result in negative economic effects.
This study detected 30 (12.5%) Salmonella isolates in 240 chickens of different ages. This result is consistent with that reported by Ibrahim et al.25 and El-Fakar and Rabie26. Although a study by Antunes et al.27 showed a higher Salmonella incidence in chickens, it concerned poultry farms in countries that have different surveillance rates and biosecurity levels for poultry farms.
The isolates in this study were categorized antigenically and serologically into 5 serogroups with characteristic antigenic properties based on O and H antigens. Salmonella Enteritidis (11), Salmonella Typhimurium (8) and Salmonella Muenster (6) were the most prevalent serotypes followed by Salmonella Anatum (3) and Salmonella Virchow (2) (Table 2). These results supported those obtained by Fashae et al.28, Muhammad et al.29, Suresh et al.30, Abd El-Ghany et al.31 and Ahmed and Shimamoto32.
The molecular detection of Salmonella species using PCR showed that there were 5 additional isolates that were typical for Salmonella by conventional (culture, biochemical and serological) methods. This result suggests that the PCR technique would be a sensitive, rapid and specific diagnostic tool for Salmonella detection on poultry farms33.
The dissemination of antibiotic resistance genes and emergence of antibiotic resistant Salmonella serovars reflects the worldwide interest and public health concerns about these pathogens, especially in Africa and Asia34,35. Multidrug resistance, particularly in Enterobacteriaceae, represents a significant public health concern in both developing and developed countries36. Hence, the assessment of antibiotic resistance of Salmonella species isolated from chickens has become an important integrated process in pathogen control methods. Antibiotic susceptibility results of the current study revealed three antibiotic profiles among the Salmonella isolates (Table 3). All Salmonella isolates were phenotypically susceptible to ciprofloxacin and chloramphenicol as was reported by Murugkar et al.37, Salehi et al.38 and Begum et al.39. In contrast, Agbaje et al.40 and Muthu et al.41 showed that the majority of isolated Salmonella serovars were resistant to ciprofloxacin and chloramphenicol. This difference may be due to the continuous use of antibiotics in a given locality that leads to the emergence of resistant strains of the same pathogens.
Pathogenicity islands include large clusters of genes that facilitate Salmonella colonization, invasiveness and establishment of systemic infection in the host. They also facilitate acquisition of a single island that can convert a non-pathogenic microorganism to a pathogenic one2,13-19. In the current study the virulence genes for five major SPIs from the three major predominate Salmonella serovars: S. Enteritidis, S. Typhimurium and S. Muenster were examined and found that the five major pathogenicity islands (SP-1-SP-5) genes were present in all of them (Fig. 1), which is concurrent with studies by Soto et al.23 and Sanchez-Jimenez et al.24. The results also revealed that all three predominate serovars, but particularly S. Enteritidis and S. Typhimurium, were present in our samples. These two serovars are pathogenic to both humans and chickens and cause 80% of Salmonella infections in humans.
CONCLUSION
There are several different Salmonella serotypes, including Salmonella enteritidis, Salmonella typhimurium, Salmonella muenster, Salmonella anatum and Salmonella virchow circulating in chicken farms in Egypt. Among these, Salmonella enteritidis and Salmonella typhimurium were the most prevalent serotypes. These isolates represent a public health risk and also exhibit several antibiotic resistant patterns as well as the most common pathogenicity islands.
SIGNIFICANCE STATEMENTS
This study identified prevalent multidrug resistant Salmonella species among chickens in poultry farms in Egypt. These serovars carry five major virulence associated genes and represent potential public health hazards. As such, measures to control the prevalence of Salmonella in poultry farms are urgently needed. This study examined in greater detail the pathogenicity of Salmonella species in an agricultural setting and revealed critical areas where Salmonella control measures have been unsuccessful. These results may provide insight into antibiotic susceptibility and virulence-associated factors of Salmonella in poultry farms and form a basis for developing more successful control measures.